Peptide composition and uses thereof

ABSTRACT

Subject of the invention is a composition comprising at least one fragment of the peptide ESAT-6 and at least one fragment of the peptide CFP-10. Preferably, the fragments comprise at least two sets of peptides, a first set comprising at least one peptide of from about 7 to 14 amino acid residues in length and a second set comprising at least one peptide of from 16 amino acid residues or greater. The invention also relates to diagnostic methods using the composition.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of European Patent Application No.EP14190111.6, filed Oct. 23, 2014, which application is incorporated byreference herein in its entirety.

BACKGROUND OF THE INVENTION

The invention relates to the field of immunological-based diagnosticassays including an assay to measure cell-mediated immunoresponsiveness.

Immunological-based diagnostic assays are important tools in detecting avariety of disease conditions. The effectiveness of these types ofassays lies in part in the specificity of components within the immunesystem. Notwithstanding this specificity, immunological-baseddiagnostics are not necessarily always sensitive enough to detect lowgrade infection or the presence of a persistent low level infection orin subjects with active or latent infectious disease states. There is aneed to develop diagnostic assays with enhanced sensitivity in relationto cell-mediated immunoresponsiveness.

One form of immunological-based diagnostic assay involves thestimulation of T-cells within antigens or mitogens in either isolatedcell culture or in whole blood culture followed by the detection ofeffector molecules such as cytokines produced by the stimulated T-cells(also referred to as effector T-cells). The effector molecules aregenerally detected using techniques such as enzyme immunoassays,multiplex bead analysis, ELISpot and flow cytometry. However, suchassays are often not sensitive and selective enough for routinediagnostic application.

The ability to quickly assess cell-mediated immunity and with a highdegree of sensitivity is of clinical importance. This is particularlythe case with immune system compromised patients. A clinician needs tohave an appreciation of the development of a disease state and itseffect on the host's immune system.

WO2013/000021 A1 discloses an improved cell mediated immune responseassay with enhanced sensitivity. The method for measuring cell-mediatedimmune response activity in a subject comprises contacting lymphocytesfrom the subject with at least two sets of peptides, a first setcomprising at least one peptide of from about 7 to 14 amino acidresidues in length and a second set comprising at least one peptide offrom 16 amino acid residues or greater which peptides encompass all orpart of a protein antigen. Various protein antigens have been describedin the art which induce a cellular immune reaction. For example,WO95/01441 A1 discloses a peptide antigen ESAT-6 isolated frommycobacteria. WO97/09429 A2 discloses a peptide antigen CFP-10 isolatedfrom mycobacteria.

Laurens et al. (Clin. and Diagn. Lab. Imm., March 2000, Vol. 7 No. 2,p155-160) describe a method of diagnosing tuberculosis based on ESAT-6and CFP-10. They find that a combination of both antigens increasesensitivity and specificity of in vitro and in vivo diagnostic assayswhen compared to use the isolated antigens.

WO2008/141226 discloses bacterial expression systems for variousantigens, such as CFP-10 and ESAT-6. The document does not relate tofragments of the antigens.

WO2009/024822 discloses a list of antigens for use in a vaccine, butdoes not disclose or suggest specifically combining CFP-10 and ESAT-10or fragments thereof.

WO2014/085713A1 relates to methods and compositions for detectingtuberculosis based on marker proteins. A diagnostic assay is describedin which large fragments of ESAT-6 and CFP-10 are obtained in a probe bytrypsin digestion. The document does not disclose defined sets ofpeptides or methods in which such peptides interact with lymphocytecells.

However, there is an ongoing need for novel sensitive and selectiveantigen compositions and corresponding immunological-based diagnosticassays.

PROBLEM UNDERLYING THE INVENTION

The problem underlying the invention is to provide novel sensitive andselective antigen compositions and corresponding immunological-baseddiagnostic assays. Further, the problem is to provide an improvedcomposition and assay for diagnosing tuberculosis.

DISCLOSURE OF THE INVENTION

Surprisingly, it was found that the problem underlying the invention isovercome by compositions and methods according to the claims. Furtherembodiments of the invention are outlined throughout the description.

Subject of the invention is a composition comprising at least onefragment of ESAT-6 and at least one fragment of CFP-10.

As used herein, a “fragment” is a peptide having less amino acids thanfull length ESAT-6 or CFP-10.

The inventive composition comprises fragments of ESAT-6 and CFP-10.However, it is not excluded that other fragments or antigens arepresent. In an embodiment of the invention, no other fragments and/orantigens are present. In this embodiment, the immunogenic peptides inthe composition are all fragments of ESAT-6 and CFP-10.

In a preferred embodiment, the fragments comprise at least two sets ofpeptides, a first set comprising at least one peptide of from about 7 to14 amino acid residues in length and a second set comprising at leastone peptide of from 16 amino acid residues or greater.

In this embodiment, the first set of peptides of from about 7 to 14amino acid residues in length could be regarded as “the short peptides”,whereas the second set comprising at least one peptide of from 16 aminoacid residues or greater could be regarded as “the long peptides”.

In a preferred embodiment, the peptides encompass part of the proteinantigen ESAT-6 and CFP-10. In an embodiment, the first and the secondset of peptides each comprise fragments of ESAT-6 and CFP-10.

According to the invention, it is not excluded that other fragments ofESAT-6 and CFP-10 are part of the composition. Thus, fragments of ESAT-6and CFP-10 may also be present having a length of up to 6 amino acids,or 15 amino acids.

In a preferred embodiment, no full length ESAT-6 and/or CFP-10 are partof the composition. In other embodiments, no fragments of ESAT-6 and/orCFP-10 are included, which have a length of more than 75, more than 50or more than 25 amino acids.

ESAT-6 (UniProt accession no: P0A564; Entrez accession no: 886209) is a6 kDa early secretary antigenic target of M. tuberculosis. WO95/01441 A1discloses the protein sequence and a method for isolation frommycobacteria.

CFP-10 (UniProt accession no: P0A566; Entrez accession no: 886194) is a10 kDa protein also known as ESAT-6-like protein eesxB and secretedantigenic protein MTSA-10. WO97/09429 A2 discloses the sequence and amethod for isolating CFP-10 from mycobacteria.

Laurens et al. (Clin. and Diagn. Lab. Imm., March 2000, Vol. 7 No. 2,p155-160) describe a method of diagnosing tuberculosis based on ESAT-6and CFP-10. They find that a combination of both antigens increasesensitivity and specificity of in vitro and in vivo diagnostic assayswhen compared to use the isolated antigens. However, the prior art hasnot described an inventive composition, which combines fragments ofESAT-6 and CFP-10 for inducing cellular immune response.

By “about 7 to 14 amino acids” means 7, 8, 9, 10, 11, 12, 13 or 14 aminoacids. This is considered herein a first set of peptides.

By “greater than 15 amino acids” means from 16 to the entire length ofthe protein antigen, including from 16 to 50 amino acids. This isconsidered a second set of peptides. The present method is not to belimited to which set of peptides is referred to as first or second. Eachset comprises from at least one peptide to a series of overlappingpeptides. The lengths of the “long peptides” having 16 amino acids ormore are limited by the lengths of ESAT-6 (95 amino acids) and CFP-10(100 amino acids). In specific embodiments, the maximum lengths of thelong peptides are below 90, below 75, below 50 or below 30 amino acids.The peptides can be obtained by known means, for example recombinantpeptide production methods in vitro or in vivo, or by digestion ofESAT-6, CFP-10 or derivatives thereof.

The co-incubation of the 7 to 14 amino acid peptides and the greaterthan 15 amino acid peptides derived from the protein antigen with thelymphocytes results in a more sensitive assay, enabling earlierdetection of lymphocyte stimulation than would otherwise be possible.The increased sensitivity includes at least a 10% increased detection ofeffector molecules compared to co-incubation with a single peptide inthe 7 to 14 amino acid range or >15 amino acid range derived from theantigen or the whole antigen itself. The ability to increase thesensitivity of a cell-mediated immune response assay also enables lesssensitive means of detection of effector molecules. Furthermore, themagnitude of the cell-mediated immune response detected in the assaypresently disclosed can be correlated to the disease state, progressionand/or severity. Hence, the present disclosure teaches an assay of acell-mediate immunoresponsiveness in a subject.

Without limiting the present invention to any one theory or mode ofaction, the two sets of peptides, the 7 to 14mer peptides and >15merpeptides enables detection by both CD4⁺ and CD8⁺ T-cells. The CD4⁺T-cells recognize the >15mer peptides and CD8⁺ T-cells recognize the 7to 14mer peptides. These peptides may be referred to herein as “CD4⁺peptides” (>15mer peptides) or “CD8⁺ peptides” (7 to 14mer peptides).

Preferably, the composition comprises multiple peptides of from about 7to 14 amino acid residues and multiply different peptides of from 16amino acid residues or greater. In this respect, “multiple peptides”refers to a pool, which may comprise for example from 2 to 2000,preferably from 10 to 1000 or from 20 to 500 different peptides. Whenmultiple peptides are comprised, the immune response is often improved.

In a preferred embodiment, the composition further comprises at leastone sugar. Preferably, the sugar is a non-reducing sugar, mostpreferably trehalose.

It has been described in the art, for example WO2004/042396A1, thatsugars may enhance the sensitivity of such methods and compositions.

In a preferred embodiment, the sugar is a non-reducing sugar. A“non-reducing sugar” in particular refers to a sugar which does notreact with a detection reagent for reducing sugars, such as Fehling'ssolution, Benedict's reagent or Tollens' reagent. A non-reducing sugardoes not comprise a free reducing end and accordingly, does not comprisea free aldehyde or free ketone group. The non-reducing sugar may haveany length and may be linear or branched. In certain embodiments, thenon-reducing sugar comprises at least two monosaccharide units.According to one embodiment, in any and all of the monosaccharide unitsof the non-reducing sugar the carbon atoms neighboring the oxygen atomin the ring structure do not comprise a hydroxyl group and thus, do notcomprise an anomeric hydroxyl group. According to one embodiment, thering structures of the monosaccharide units of the non-reducingoligosaccharide do not comprise a hemiacetal or hemiketal group.According to one embodiment, the non-reducing sugar is anoligosaccharide which comprises 10 monosaccharide units or less, morepreferably 8 monosaccharide units or less, 6 monosaccharide units orless, 5 monosaccharide units or less, 4 monosaccharide units or less, 3monosaccharide units or less or 2 monosaccharide units. Preferably, thenon-reducing sugar is a disaccharide. According to one embodiment, theglycosydic bonds are formed between the monosaccharide units byattaching the reducing end of one monosaccharide unit to the reducingend of another monosaccharide unit. Preferred examples of thenon-reducing sugar are sucrose and trehalose. Trehalose is particularlypreferred because experiments show that trehalose increases themagnitude of response and thus may increase the assay sensitivity andfurthermore, a composition comprising trehalose and an antigen showsexcellent storage stability. However, the non-reducing sugar can also bea monosaccharide, wherein, e.g., the reducing end is coupled to andthereby blocked by another chemical entity. Accordingly, thenon-reducing sugar may be derivatized. Examples of sugar derivatives areaminosugars wherein one or more hydroxyl group is substituted by anamino group or an acetylamino group. In preferred embodiments, thenon-reducing sugar is not substituted and in particular is notderivatized. According to one embodiment, the non-reducing sugar is nota polysaccharide. In certain embodiments, the non-reducing sugar is notbound to a protein, peptide or lipid or other macromolecule. Accordingto one embodiment, the non-reducing sugar is not comprised in a cellculture medium or other medium. According to one embodiment, thenon-reducing sugar is not comprised in a liquid. The non-reducing sugaris metabolizable by immune cells comprised in the sample. According toone embodiment, the non-reducing sugar is a non-reducing sugar whichwhen present in an appropriate concentration in the incubationcomposition comprising the sample and the antigen is capable ofincreasing the release of interferon gamma by re-stimulated T-cells.

The sugar can be part of the composition. Alternatively, the sugar canbe added when the composition is contacted with the lymphocytes.

According to one embodiment, the non-reducing sugar is a non-reducingdisaccharide, preferably being selected from trehalose and sucrose. Theconcentration of the non-reducing sugar in the incubation composition isaccording to one embodiment at least 1.5 mg/ml, preferably at least 2mg/ml. Suitable ranges for the concentration of the non-reducing sugarin the incubation composition are also described above and it isreferred to the above disclosure.

The composition of the invention can be used as a diagnostic ortherapeutic composition. Such uses and applications are outlined furtherbelow.

The composition and method of the invention are especially applicablefor determining a cell-mediated immune response, preferably as adiagnostic composition or in a diagnostic method.

The composition and method of the invention are especially applicablefor diagnosing tuberculosis. Assay for diagnosing tuberculosis withESAT-6, CFP-10 or a combination thereof have been described in the art.

A method for measuring cell-mediated immune response activity in asubject is therefore provided herein, the method comprising contactinglymphocytes from the subject with a composition of claim 1 and measuringthe presence or elevation in the level of an immune effector moleculefrom immune cells wherein the presence or level of the immune effectormolecule is indicative of the level of cell-mediated responsiveness ofthe subject to the antigen.

Preferably, the subject is a human and the sample is undiluted wholeblood. Alternatively, the sample is whole blood which comprises fromabout 10% to 100% by volume of the sample to be assayed or comprisesfrom about 50% to 100% by volume of the sample to be assayed orcomprises from about 80% to 100% by volume of the sample to be assayed.The sample volume may be in microliter or milliliter amounts such asfrom 0.5 μl to 5 ml. Conveniently, the whole blood is collected in atube comprising heparin and the immune effector molecule is IFN-γ.Generally, the immune effectors are detected with antibodies specificfor same such as using EL1SA or an ELISpot.

One or more further additives can be added and thus be included in thecomposition. E.g. one or more additives can be added that are necessaryor advantageous for sample preparation and/or sample preservation suchas e.g. a suitable anticoagulant if the sample is a blood sample.Preferably, the anticoagulant is heparin. Additives should not becomprised in a concentration wherein they could interfere with thecell-mediated immune response. According to one embodiment, no simplesugar is added to the incubation composition in addition to thenon-reducing sugar. According to one embodiment, no reducing sugar, inparticular no reducing monosaccharide is added to the incubationcomposition in addition to the non-reducing sugar. The subject may havean infection by a pathogenic agent selected from Mycobacterium speciessuch as Mycobacterium tuberculosis or tuberculosis (TB), Staphylococcusspecies, Streptococcus species, Borrelia species, Escherichia coli,Salmonella species, Clostridium species, Shigella species, Proteusspecies, Bacillus species, Herpes virus, Hepatitis B or C virus andHuman immune deficiency virus (HIV) or a disease resulting therefrom.

A method is also provided of allowing a user to determine the status ofcell-mediated immunoresponsiveness of a subject, the method including:

(a) receiving data in the form of levels or concentrations of an immuneeffector molecule which, relative to a control, provide a correlation asto the state of cell-mediated immunoresponsiveness in a subject, via acommunications network, the immune effector molecule measured afterexposure of lymphocytes to a composition of the invention;

(b) processing the data via univariate or multivariate analysis toprovide an immunoresponsiveness value;

(c) determining the status of the subject in accordance with the resultsof the immunoresponsiveness value in comparison with predeterminedvalues; and

(d) transferring an indication of the status of the subject to the uservia the communications network.

In an embodiment, the composition comprises fat least one additionalantigen or fragments thereof. Preferably, the additional antigen is atuberculosis antigen, preferably TB7.7 or TB37.6.

In an embodiment, the lymphocytes are contacted with a combination ofCD4⁺ and CD8⁺ peptides.

The terms “T-cells” and “T-lymphocytes” are used interchangeably herein.An “immune cell” includes a lymphocyte such as a T-cell.

A “combination” also includes multi-part such as a two-part compositionwhere the agents are provided separately and used or dispensedseparately or admixed together prior to dispensation. For example, amulti-part assay pack may have a series of overlapping peptides fromabout 7 to 14 amino acid residues in length and/or greater than 15 aminoacid residues in length which encompass all or part of a protein antigenagainst which a cell-mediated immune response is to be measured. Hence,this aspect of the present disclosure includes agents dried and loose orimmobilized to a compartment wall or solid support in an assay pack.

The present disclosure contemplates sets of peptides. The term “set” maybe replaced by other terms such as “pool”, “group”, “series”,“collection” and the like without departing from the method instantlydisclosed. Each set comprises at least one peptide and includes in anembodiment a series of overlapping peptides. Hence, a first set maycontain a series of overlapping peptides of from 7 to 14 amino acidresidues in length. These peptides are recognized by CD8⁺ T-cells, (CD8⁺peptides). A second set may contain a series of overlapping peptides ofgreater than 15 amino acid residues in length. These peptides arerecognized by CD4⁺ T-cells (CD4⁺ peptides) Both sets of peptidesencompasses the entire length of or part of a protein antigen.Furthermore, the peptides do not necessarily have to be overlapping ormay overlap by a single amino acid or multiple amino acids.

Reference to a series of overlapping peptides from about 7 to 14 aminoacid residues in length which encompass all or part of a protein antigenmeans a peptide of from about 7 amino acid residues in length to amaximum of 14 amino acid residues which in total span from every aminoacid residues which in total span amino acid residues to up to 6 aminoacid residues of a protein antigen from its N-terminal end to itsC-terminal end or part thereof. Hence, if the length of a given peptideis x amino acid residues in length wherein x is from about 7 to 14, thenthe extent of overlap between two consecutive peptides is from x-1 tox-6. In an embodiment, the overlap of each consecutive peptide is x-1. Aseries of overlapping peptides of greater than 15 amino acid residues inlength also spans all or part of a protein antigen wherein each peptideis at least 16 amino acid residues in length or up to the length of thefull protein antigen. In an embodiment, a peptide of greater than 15amino acid residues in length is from 16 to 50 amino acids such as 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 aminoacid residues. As indicated above, there is no necessity for thepeptides to overlap provided there is at least one set of one or more 7to 14 amino acid peptides and another set of at least one >15merpeptides.

The present disclosure includes the case where each peptide in theseries is the same length (i.e. x). However, the series of peptides maycomprise a mixture of X₁, x₂, x_(j) . . . x_(i) peptides where each ofx_(i) peptides is from about 7 to 14 amino acid residues in length orgreater than 15 amino acid residues in length.

Enabled herein is a method for detecting a cell-mediated immune responsein a subject, the method comprising incubating lymphocytes from thesubject with an inventive composition and then screening for levels ofeffector molecules produced by activated lymphocytes.

Lymphocytes are activated by co-incubation with at least two sets ofpeptides, a first set comprising at least one peptide of from about 7 to14 amino acid residues in length and a second set comprising at leastone peptide of from 16 amino acid residues or greater which peptidesencompass all or part of a protein antigen.

The present disclosure teaches augmentation of production of effectormolecules from lymphocytes exposed to an inventive composition. Suchlymphocytes are “activated” or “stimulated” lymphocytes. Theaugmentation occurs by exposing the cells to an inventive composition.The level of the response is greater than in the presence of wholeantigen or a peptide derived from the antigen which is less than 7 aminoacids or greater than 14 amino acids. This enables a more sensitiveassay in order to assess the cell-mediated immune responsiveness of asubject. The present disclosure, therefore, enables an assay to detect,assess or otherwise monitor a cell-mediated response in a subject bymeasuring the presence or level of effector molecules from T-cellsstimulated by an inventive composition. The assay also enables earlierdetection of cell-mediated responsiveness. In an embodiment, the assaytaught therein enhances the sensitivity of a cell-mediated assay whichmay enable less sensitive detection assays to be employed. Furthermore,the extent or magnitude of the cell-mediated immune response is proposedto be reflective or informative of the state, progression and/orseverity of a disease condition. For example, the magnitude of theresponse may determine if a subject has a latent or active or acuteinfection or disease condition.

Conveniently, the CD4⁺ and/or CD8⁺ peptides are divided into separatepools of peptides.

Without limiting the present invention to any one theory or mode ofaction, at least two sets of peptides enables both CD4⁺ and CD8⁺epitopes to be stimulated. The peptides may be referred to herein as“CD4⁺ peptides” (>15mer peptides) or “CD8⁺ peptides” (7 to 14merpeptides).

An additional agent may also be added to the incubation mixture such asto modulate the activity of regulatory T-cells (T-reg cells). The latterencompasses inhibiting the suppressor function of T-reg cells. Agentswhich modulate T-reg cells encompassed herein include a CD25 ligand; asense or antisense oligonucleotide to genetic material encoding JAK1 orTYK2; a neutralizing antibody; a CpG containing oligonucleotide; anoligonucleotide acting as a toll-like receptor (TLR) modulating agent;and other TLR modulating agents.

In a particular embodiment, the T-reg cells are immune responsesuppressor cells the activity of which is inhibited.

A “CpG molecule” means an oligonucleotide comprising a CpG sequence ormotif.

The present disclosure provides a means to determine the responsivenessof cell-mediated immunity in a subject and, in turn, teaches thedetermination of whether a disease condition or an agent induces or isassociated with immunosuppression. The method also enables diagnosis ofinfectious diseases, pathological conditions, determination of the levelof immunocompetence and assessing of immune cell responsiveness toendogenous or exogenous agents as well as assessing exposure to proteintoxicants. The assay also enables screening of subjects previouslyexposed to a particular antigen, such as an antigen associated with adisease, infection or contaminant.

Accordingly, an aspect taught herein contemplates a method for measuringcell-mediated immune response activity in a subject, the methodcomprising contacting lymphocytes from the subject with an inventivecomposition and measuring the level of an immune effector moleculeproduced by immune cells wherein the level of the immune effectormolecule is indicative of the level of cell-mediatedimmunoresponsiveness of the subject.

Another aspect contemplated herein is a method for measuringcell-mediated immune response activity in a subject, the methodcomprising contacting lymphocytes from the subject with an inventivecomposition and measuring the elevation in the level of an immuneeffector molecule from immune cells wherein the level of the immuneeffector molecule is indicative of the level of cell-mediatedresponsiveness of the subject wherein the level of responsiveness isindicative of the presence or absence or level or stage of a disease orcondition selected from the list comprising an infection by a pathogenicagent, an autoimmune disease, a cancer, an inflammatory condition andexposure to a toxic proteinaceous agent.

Yet another aspect enabled herein is a method for measuringcell-mediated immune response activity in a subject, the methodcomprising contacting lymphocytes from the subject with an inventivecomposition and measuring the elevation in the level of an immuneeffector molecule from immune cells wherein the level of the immuneeffector molecule is indicative of the level of cell-mediatedresponsiveness and is indicative of the presence or absence or level orstage of a disease or condition selected from the list comprising aninfection by a pathogenic agent, an autoimmune disease, a cancer, aninflammatory condition and exposure to a toxic proteinaceous agent.

Still another aspect taught by the present disclosure is an assay todetect the presence, absence, level or stage of a disease or conditionin a subject, the method comprising contacting lymphocytes from thesubject with an inventive composition and measuring the elevation in thelevel of an immune effector molecule from immune cells wherein the levelof the immune effector molecule is indicative of the disease orcondition.

The present disclosure further contemplates a method for determiningwhether an agent induces immunosuppression in a subject, the methodcomprising contacting lymphocytes from the subject after exposure to theagent with an inventive composition and measuring the presence and levelof an effector molecule from the lymphocytes wherein the level of theeffector molecule determines the level of immunosuppression induced bythe agent. In accordance with this aspect, the agent may be a medicamentor an environmental toxicant.

In an embodiment, the lymphocytes are comprised within a blood sample.In an embodiment, the blood sample is co-stimulated with an inventivecomposition.

A use is also provided for an inventive composition in the manufactureof a diagnostic assay of cell-mediated immune responsiveness by themethod of incubating lymphocytes with a limiting amount of the agonistand detecting the presence or elevation in an effector molecule.

In another embodiment, taught herein is a method for detecting whether adisease condition is inducing immunosuppression in a subject the methodcomprising contacting lymphocytes from the subject with a diseasecondition with an inventive composition and measuring the presence orlevel of an immune effector molecule from the lymphocytes wherein thelevel of the immune effector molecule is indicative of the extent ofimmunosuppression induced or associated with the disease condition.

A use is also provided for an inventive composition in the manufactureof a diagnostic assay of cell-mediated immune responsiveness. Generally,the method comprising incubating lymphocytes with an inventivecomposition.

This use includes the use for detecting or monitoring the presence,absence, level or stage of a disease or condition such as an infectionby a pathogenic agent, an autoimmune disease, a cancer, an inflammatorycondition and/or exposure to a medicament or a toxic proteinaceous agentsuch as an environmental toxicant. Measuring “an immune effectormolecule” includes measuring one or more different types of molecules.

The present disclosure further enables a method for measuringcell-mediated immune response activity in a subject, the methodcomprising contacting a regulatory T-cell from the subject with an agentselected from (i) an inhibitor of suppressor regulatory T-cells; and(ii) an activator of immune augmenting cells or a subset thereof; andfurther contacting T-cells with an inventive composition and measuringthe elevation in the level of an immune effector molecule from immunecells wherein the level of the immune effector molecule is indicative ofthe level of cell-mediated responsiveness of the subject.

Examples of inhibitors or modulators of T-reg function include CD25ligands such as but not limited to a polyclonal or monoclonal antibodyto CD25 or an antigen-binding fragment thereof, humanized or deimmunizedpolyclonal or monoclonal antibodies to CD25 or a recombinant orsynthetic form of the polyclonal or monoclonal antibodies. Otherexamples of agents include sense or antisense nucleic and moleculesdirected to the mRNA or DNA (i.e. genetic material) encoding JanusTyrosine Kinase 1 (JAK1) or Tyrosine Kinase 2 (TYK2) or small moleculeinhibitors of JAK1 or TYK2 proteins. Reference to “small molecules”includes immunoglobulin new antigen receptors (IgNARs) as described inInternational Patent Publication No. WO 2005/1 18629. Yet still furtherexamples of suitable agents stimulating agents such as CpG moleculeswhich act via Tolllike receptors (TLRs) and/or other mechanisms. Hence,CpG containing oligonucleotides and an oligonucleotide acting as a TLRmodulating agent also form part of the present disclosure.

A single type of agent may be used or two or more types of agents may beemployed to modulate T-reg cells. For example, the assay may beconducted with a CD25 ligand and a JAK1/TYK2 sense or antisenseoligonucleotide; a CD25 ligand and a TLR modulating agent; a JAK1/TYK2sense or antisense oligonucleotide and a TLR modulating agent; or a CD25ligand, a JAK1/TY 2 sense or antisense oligonucleotide and a TLRmodulating agent. Alternatively, just one type of agent is employed. Inanother alternative, a CpG comprising oligonucleotide and a TLRmodulating agent is used.

Reference to a “subject” includes a human or non-human species includingprimates, livestock animals (e.g. sheep, cows, pigs, horses, donkey,goats), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs,hamsters), companion animals (e.g. dogs, cats), avian species (e.g.poultry birds, aviary birds), reptiles and amphibians. The presentsubject matter has applicability in human medicine as well as havinglivestock and veterinary and wild-life applications which includes thehorse, dog and camel racing industries. For example, the assay of thepresent disclosure may be routinely carried out on horses before and/orafter heavy exertion (such as a race) to screen for evidence ofexercise-induced pulmonary hemorrhage (EIPH). All horses exhibit someform of EIPH to some degree during exercise. However, sub-clinical formsof EIPH can be hard to detect.

Reference to a “human” includes particular populations of humans such aspediatric, elderly and infirmed populations of humans as well asparticular cohorts or populations of humans of a particular ethnicity.

In another embodiment, the subject is a human and the cell-mediatedimmune response assay is used in screening for responsiveness topathogenic microorganisms, viruses and parasites, potential fordevelopment or monitoring autoimmune conditions, Celiac's disease,monitoring a subject's response to oncological challenge and fordetermining the presence of any immunodeficiency or immunosuppression.The latter may occur, for example, due to certain medicaments includingvarious chemotherapeutic agents. Alternatively, exposure toenvironmental proteinaceous toxicants and pollutants.

The immune effector molecules may be any of a range of molecules whichare produced in response to cell activation or stimulation by anantigen. Although an interferon (IFN) such as IFN-γ is a particularlyuseful immune effector molecule, others include a range of cytokinessuch as interleukins (IL), e.g. IL-2, IL-4, IL-6, IL-6 (CXCL8), IL-10,IL-12, IL-13, IL-16 (LCF) or IL-17, IL-la (IL-1F1), IL-I β (IL-1 F2),IL-Ir (IL-1F3), Tumor Necrosis Factor alpha (TNF-α), Transforming GrowthFactor beta (TGF-β), a Colony Stimulating Factor (CSF) such asGranulocyte (G)-CSF or Granulocyte Macrophage (GM)-CSF, complementcomponent 5a (C5a), Groa (CXCL1), sICAM-1 (CD54), IP-10 (CXCL10), I-TAC(CXCL11), MCP-1 (CCL2), MIF (GIF), MIP-Ia (CCL3), MIP-Iβ (CCL4), RANTES(CCL5) or MIG (CXCL9).

The present disclosure also enables a method for measuring cell-mediatedimmune response activity in a subject, the method comprising contactinglymphocytes from the subject with an inventive composition and measuringthe level of an immune effector molecule from immune cells wherein thelevel of the immune effector molecule is indicative of the level ofcell-mediated responsiveness of the subject.

The assay taught herein enables detection of the presence or absence orlevel or stage of a disease or condition in a subject such as infectionby a pathogenic agent, an autoimmune disease, cancer, exposure to aninflammatory agent exposure to a medicament, exposure to a toxicproteinaceous agent and immunodeficiency or immunosuppression such asinduced by a disease condition.

In an embodiment, the sample collected from the subject is generallydeposited into a blood collection tube. A blood collection tube includesa blood draw tube or other similar vessel. Conveniently, when the sampleis whole blood, the blood collection tube is heparinized. Alternatively,heparin is added to the tube after the blood is collected.

Notwithstanding that whole blood is particularly contemplated and a mostconvenient sample, the present disclosure extends to other samplescontaining immune cells such as lymph fluid, cerebral fluid, tissuefluid and respiratory fluid including nasal and pulmonary fluid as wellas samples having undergone cell depletion. Reference to “whole blood”includes whole blood which has not been diluted such as with tissueculture, medium, reagents, excipients, etc. In one embodiment, the term“whole blood” includes an assay sample (i.e. reaction mixture)comprising at least 10% by volume whole blood. Additional agents may beadded such as culture media, enzymes, excipients antigen and the likewithout departing from the sample comprising “whole blood”.

Blood volumes may be from about 0.5 μl to 200 ml. The present disclosurealso enables the use of acoustic microstreaming to improve the mixing ofcomponents in the assay. Acoustic microstreaming is disclosed inInternational Patent Application No. PCT/AU01/00420.

Hence, contemplated herein is a method of mixing one or more lymphocytesand an inventive composition in a vessel, the method comprisingproviding from about 0.5 μl to 150 μl of fluid comprising the componentsin the vessel so as to establish a discontinuity in acoustic impedanceand applying an acoustic signal to cause mixing within the fluid. Asecond acoustic signal may also be applied, the first and second signalshaving respective frequencies each selected from about 1 Hz to about20,000 Hz in an alternating manner to effect chaotic mixing within thefluid.

The use of blood collection tubes is compatible with standard automatedlaboratory systems and these are amenable to analysis in large-scale andrandom access sampling. Blood collection tubes also minimize handlingcosts and reduce laboratory exposure to whole blood and plasma and,hence, reduce the risk of laboratory personnel from contracting apathogenic agent such as HIV or Hepatitis B virus (HBV) or Hepatitis Cvirus (HCV).

Combining the incubation step with the collection tube is particularlyefficacious and enhances the sensitivity of the assay as does theoptional feature of incubating the cells in the presence of a simplesugar such as dextrose or glucose.

The cells of the cell-mediated immune system lose the capacity to mountan immune response in whole blood after extended periods following blooddraw from the subject, and responses without intervention are oftenseverely reduced or absent 24 hours following blood draw. The reductionof labor and need for specialized plastic ware allows cell-mediatedimmune stimulation with the peptide antigens to be performed at thepoint of care locations such as physicians' offices, clinics, outpatientfacilities and veterinary clinics or on farms. Once antigen stimulationis complete, the requirement for fresh and active cells no longerexists. IFN-γ and other cytokines or immune effector molecules arestable in plasma and, thus, the sample can be stored, or shipped withoutspecial conditions or rapid time requirements.

The incubation step may be from 1 to 50 hours, such as 1 to 40 hours or8 to 24 hours. A period of 24 hours is particularly convenient.

The ability to measure cell-mediated immunity is important for assessinga subject's ability to respond to an infection by a pathogenic agentsuch as a microorganism or virus or parasite, to mount an autoimmuneresponse such as in autoimmune diabetes or to protect against cancers orother oncological conditions or to detect an inflammatory condition orto detect exposure or sensitivity of a subject to a toxic agent such asberyllium. The assay described herein also enables detection of diseaseconditions which lead to immunosuppression or immunosuppression inducedby medicaments Consequently, reference to “measuring a cell-mediatedimmune response in a subject” includes and encompasses immune diagnosisof infectious and autoimmune diseases, a marker for immunocompetence aswell as a marker for inflammatory diseases, cancer and toxic agents.Importantly, the combined innate and/or adaptive immune responsivenessis determined. Furthermore, the ability to use small blood volumesenables assays to be readily conducted on, for example, the pediatric,elderly and infirmed populations. The assay herein enables earlydetection or more sensitive detection of immunoresponsiveness.

In an embodiment, disease conditions leading to immunosuppressioninclude chronic infection and cancer. Medicaments which can lead toimmunosuppression include those used to treat rheumatoid arthritis,cancer and inflammatory bowel disease.

Pathogenic or infectious agents include bacteria, parasites and viruses.Examples of bacteria include Gram positive and Gram negativemicroorganisms such as Mycobacterium species, Staphylococcus species,Streptococcus species, Escherichia coli, Salmonella species, Clostridiumspecies, Shigella species, Proteus species, Bacillus species, Hemophilusspecies, Borrelia species amongst others. Mycobacterium tuberculosis isa particularly useful target as well as conditions arising frominfection by M. tuberculosis such as tuberculosis (TB). Examples ofviruses include Hepatitis virus (Hepatitis B virus and Hepatitis Cvirus), Herpes virus and Human immune deficiency virus (HIV) as well asdiseases resulting therefrom. Parasites include Plasmodium species,ringworm, liver parasites and the like. Other pathogenic agents includeeukaryotic cells such as yeasts and fungi.

In an embodiment, the additional tuberculosis antigen is TB7.7 orTB37.6. In an embodiment, the subject is infected with HIV.

The present invention is particularly useful for screening for exposureto M. tuberculosis. Hence, the present disclosure teaches a method formeasuring cell-mediated immune response activity in a subject, themethod comprising contacting lymphocytes from the subject with aninventive composition, wherein the fragments optionally comprisepeptides from an additional antigen is selected TB7.7 and TB37.6 fromMycobacterium tuberculosis and measuring the level of an immune effectormolecule produced by immune cells wherein the level of the immuneeffector molecule is indicative of the level of cell-mediatedimmunoresponsiveness of the subject to M. tuberculosis.

Autoimmune diseases contemplated herein for detection include inter aliaalopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease multiple sclerosis, autoimmune disease ofthe adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune oophoritis and orchitis, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatiguesyndrome (CFIDS), chronic inflammatory demyelinating, chronicinflammatory polyneuropathy, Churg-Strauss syndrome, cicatricialpemphigoid, crest syndrome, cold agglutinin disease, Crohn's disease,dermatitis herpetiformis, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia, glomerulonephritis, Grave's disease,Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulindependent diabetes (Type I), lichen planus, lupus, Meniere's disease,mixed connective tissue disease, multiple sclerosis, myasthenia gravis,myocarditis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome,rheumatic fever, rheumatoid arrthritis, sarcoidosis, scleroderma,Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus,Takayasu arteritis, temporal arteritis/gianT-cell arteritis, ulcerativecolitis, uveitis, vasculitis and vitiligo.

It is generally important to assess the potential or actualcell-mediated responsiveness in subjects exposed to these infectiousentities. The method of the present disclosure can also be used todetect the presence or absence of these conditions as well as the levelor stage of disease process.

Other disease conditions which can lead to immunosuppression includeinflammatory disease conditions.

Examples of inflammatory disease conditions contemplated by the presentdisclosure include but are not limited to those disease and disorderswhich result in a response of redness, swelling, pain, and a feeling ofheat in certain areas that is meant to protect tissues affected byinjury or disease. Inflammatory diseases which can be treated using themethods of the present disclosure include, without being limited to,acne, angina, arthritis, aspiration pneumonia, disease, empyema,gastroenteritis, inflammation, intestinal flu, NEC, necrotizingenterocolitis, pelvic inflammatory disease, pharyngitis, PID, pleurisy,raw throat, redness, rubor, sore throat, stomach flu and urinary tractinfections, chronic inflammatory demyelinating polyneuropathy, chronicinflammatory demyelinating polyradiculoneuropathy, chronic inflammatorydemyelinating polyneuropathy, chronic inflammatory demyelinatingpolyradiculoneuropathy. In terms of non-human applications, the presentdisclosure extends to detecting EIPH in horses and various conditions inanimals such as facial tumor disease in the Tasmanian Devil.

Cancer therapy also is somewhat dependent on cell-mediated immunity andthe cancer itself or drugs used to treat cancer can lead toimmunosuppression. Cancers contemplated herein include: a group ofdiseases and disorders that are characterized by uncontrolled cellulargrowth (e.g. formation of tumor) without any differentiation of thosecells into specialized and different cells. Such diseases and disordersinclude ABL1 protooncogene, AIDS related cancers, acoustic neuroma,acute lymphocytic leukaemia, acute myeloid leukaemia, adenocysticcarcinoma, adrenocortical cancer, agnogenic myeloid metaplasia,alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma,aplastic anaemia, astrocytoma, ataxia-telangiectasia, basal cellcarcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stemglioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoidtumors, cervical cancer, childhood brain tumors, childhood cancer,childhood leukaemia, childhood soft tissue sarcoma, chondrosarcoma,choriocarcinoma, chronic lymphocytic leukaemia, chronic myeloidleukaemia, colorectal cancers, cutaneous T-Cell lymphoma,dermatofibrosarcoma-protuberans, desmoplastic-small-round-cell-tumor,ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma,esophageal cancer, Ewing's sarcoma, extra-hepatic bile duct cancer, eyecancer, eye: melanoma, retinoblastoma, fallopian tube cancer, fanconianemia, fibrosarcoma, gall bladder cancer, gastric cancer,gastrointestinal cancers, gastrointestinal-carcinoid-tumor,genitourinary cancers, germ cell tumors,gestational-trophoblastic-disease, glioma, gynaecological cancers,hematological malignancies, hairy cell leukaemia, head and neck cancer,hepatocellular cancer, hereditary breast cancer, histiocytosis,Hodgkin's disease, human papillomavirus, hydatidiform mole,hypercalcemia, hypopharynx cancer, intraocular melanoma, islet cellcancer, Kaposi's sarcoma, kidney cancer, Langerhan's-cell-histiocytosis,laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lipcancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer,malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma, merkelcell cancer, mesothelioma, metastatic cancer, mouth cancer, multipleendocrine neoplasia, mycosis fungoides, myelodysplastic syndromes,myeloma, myeloproliferative disorders, nasal cancer, nasopharyngealcancer, nephroblastoma, neuroblastoma, neurofibromatosis, nijmegenbreakage syndrome, non-melanoma skin cancer,non-small-cell-lung-cancer-(NSCLC), ocular cancers, oesophageal cancer,oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovariancancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotidgland cancer, penile cancer, peripheral-neuroectodermal-tumors,pituitary cancer, polycythemia vera, prostate cancer,rare-cancers-and-associated-disorders, renal cell carcinoma,retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, salivarygland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, smallcell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma,spinal cord tumors, squamous-cell-carcinoma-(skin), stomach cancer,synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer,transitional-cell-cancer-(bladder),transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic cancer,urethral cancer, urinary system cancer, uroplakins, uterine sarcoma,uterus cancer, vaginal cancer, vulva cancer,Waldenstrom's-macroglobulinemia and Wilms' tumor.

In the above aspects, the antigen may be derived from the pathogenicagent, be associated with the disease condition or cancer or be thetoxicant. Alternatively, the infection, disease condition, cancer ortoxicant may suppress cell-mediated immunity in which case any antigento which the subject has been prior exposed could be employed.

The detection of the immune effector molecules may be measured at theprotein or nucleic acid levels. Consequently, reference to “presence orlevel” of the immune effector molecule includes direct and indirectdata. For example, high levels of cytokine mRNA are indirect datashowing increased levels of the cytokine.

Ligands to the immune effectors are particularly useful in detectingand/or quantitating these molecules. Antibodies to the immune effectorsare particularly useful. Techniques for the assays contemplated hereinare known in the art and include, for example, radioimmunoassay,sandwich assays, ELISA and ELISpot. Reference to “antibodies” includesparts of antibodies, mammalianized (e.g. humanized) antibodies,deimmunized antibodies, recombinant or synthetic antibodies and hybridand single chain antibodies. For skin tests, in humans, humanized ordeimmunized antibodies are particularly contemplated herein to detecteffector molecules.

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith the immune effector molecules or antigenic fragments thereof andeither type is utilizable for immunoassays. Methods of obtaining bothtypes of sera are well known in the art.

Polyclonal sera are less preferred but are relatively easily prepared byinjection of a suitable laboratory animal with an effective amount ofthe immune effector, or antigenic part thereof, collecting serum fromthe animal and isolating specific sera by any of the knownimmunoadsorbent techniques. Although antibodies produced by this methodare utilizable in virtually any type of immunoassay, they are generallyless favored because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlyuseful because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art.

Another aspect enabled herein, therefore, is a method for detecting animmune effector molecule in a sample comprising lymphocytes from asubject, the method comprising contacting the sample or an aliquot ofthe sample with an antibody specific for the immune effector molecule oran antigenic fragment thereof for a time and under conditions sufficientfor an antibody-effector complex to form, and then detecting the complexwherein the immune effector molecule is generated after incubation ofthe lymphocytes with an inventive composition.

A “sample” includes whole blood or a fraction thereof comprisinglymphocytes. This method includes micro-arrays, macro-arrays andnano-arrays on planar or spherical solid supports. A micro- ormacro-array is useful. A “sample” also includes a small volume sample offrom about 0.5 μl to 1000 μl including 5 μl, 10 μl, 20 μl, 50 μl and 100μl as well as larger volumes such as from 1 ml to about 200 ml such as 1ml, 2 ml, 5 ml, 10 ml or 20 ml.

A wide range of immunoassay techniques are available as can be seen byreference to U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653.

The following is a description of one type of assay. An unlabeledantibody is immobilized on a solid substrate and the sample to be testedfor the immune effector molecules (e.g. a cytokine) brought into contactwith the bound molecule. After a suitable period of incubation, for aperiod of time sufficient to allow formation of an antibody-immuneeffector molecule complex, a second antibody specific to the effectormolecule, labeled with a reporter molecule capable of producing adetectable signal, is then added and incubated, allowing time sufficientfor the formation of another complex of antibody-effector-labeledantibody. Any unreacted material is washed away, and the presence of theeffector molecule is determined by observation of a signal produced bythe reporter molecule. The results may either be qualitative, by simpleobservation of the visible signal, or may be quantitated by comparingwith a control sample containing known amounts of antigen. Thisgeneralized technique is well known to those skilled in the art as wouldbe any of a number of variations.

In these assays, a first antibody having specificity for the instantimmune effectors is either covalently or passively bound to a solidsurface. The solid surface is typically glass or a polymer, the mostcommonly used polymers being cellulose, polyacrylamide, nylon,polystyrene, polyvinyl chloride or polypropylene. The solid supports maybe in the form of tubes, beads, spheres, discs of microplates, or anyother surface suitable for conducting an immunoassay: The bindingprocesses are well known in the art and generally consist ofcross-linking covalently binding or physically adsorbing, thepolymer-antibody complex is washed in preparation for the test sample.An aliquot of the sample to be tested is then added to the solid phasecomplex and incubated for a period of time sufficient (e.g. 2-120minutes or where more convenient, overnight) and under suitableconditions (e.g. for about 20° C. to about 40° C.) to allow binding ofany subunit present in the antibody. Following the incubation period,the antibody subunit solid phase is washed and dried and incubated witha second antibody specific for a portion of the effector molecule. Thesecond antibody is linked to a reporter molecule which is used toindicate the binding of the second antibody to the effector molecule.

There are many variations to this assay. One particularly usefulvariation is a simultaneous assay where all or many of the componentsare admixed substantially simultaneously. Furthermore, binding of anantibody to a cytokine may be determined by binding of a labeledantibody directed to the first mentioned antibody.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules. Examples of suitablefluorophores are provided in Table 1. In the case of an enzymeimmunoassay, an enzyme is conjugated to the second antibody, generallyby means of glutaraldehyde or periodate. As will be readily recognized,however, a wide variety of different conjugation techniques exist, whichare readily available to the skilled artisan. Commonly used enzymesinclude horseradish peroxidase, glucose oxidase, beta-galactosidase andalkaline phosphatase, amongst others. The substrates to be used with thespecific enzymes are generally chosen for the production, uponhydrolysis by the corresponding enzyme, of a detectable color change.Examples of suitable enzymes include alkaline phosphatase andperoxidase. It is also possible to employ fluorogenic substrates, whichyield a fluorescent product rather than the chromogenic substrates notedabove. In all cases, the enzyme-labeled antibody is added to the firstantibody-antigen complex, allowed to bind, and then the excess reagentis washed away. A solution containing the appropriate substrate is thenadded to the complex of antibody-antigen-antibody. The substrate willreact with the enzyme linked to the second antibody, giving aqualitative visual signal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of antigenwhich was present in the sample. Again, the present disclosure extendsto a substantially simultaneous assay.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. The fluorescent labeled antibody is allowed to bind to thefirst antibody-antigen complex. After washing off the unbound reagent,the remaining tertiary complex is then exposed to the light of theappropriate wavelength the fluorescence observed indicates the presenceof the antigen of interest. Immunofluorescene and enzyme immunoassaytechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

There are a range of other detection systems which may be employedincluding colloidal gold and all such detection systems are encompassedby the present disclosure.

The present disclosure also contemplates genetic assays such asinvolving PCR analysis to detect RNA expression products of a geneticsequence encoding an immune effector.

In one embodiment, PCR is conducted using pairs of primers, one or bothof which are generally labeled with the same or a different reportermolecule capable of giving a distinguishable signal. The use offluorophores is particularly useful in the practice of the presentdisclosure. Examples of suitable fluorophores may be selected from thelist given in Table 1. Other labels include luminescence andphosphorescence as well as infrared dyes. These dyes or fluorophores mayalso be used as reporter molecules for antibodies.

TABLE 1 List of suitable fluorophores Probe Ex¹ (nm) Em² (nm) Reactiveand conjugated probes Hydroxycoumarin 325 386 Aminocoumarin 350 455Methoxycoumarin 360 410 Cascade Blue 375; 400 423 Lucifer Yellow 425 528NBD 466 539 R-Phycoerythrin (PE) 480; 565 578 PE-Cy5 conjugates 480;565; 650 670 PE-Cy7 conjugates 480; 565; 743 767 APC-Cy7 conjugates 650;755 767 Red 613 480; 565 613 Fluorescein 495 519 FluorX 494 520BODIPY-FL 503 512 TRITC 547 574 X-Rhodamine 570 576 Lissamine RhodamineB 570 590 PerCP 490 675 Texas Red 589 615 Allophycocyanin (APC) 650 660TruRed 490; 675 695 Alexa Fluor 350 346 445 Alexa Fluor 430 430 545Alexa Fluor 488 494 517 Alexa Fluor 532 530 555 Alexa Fluor 546 556 573Alexa Fluor 555 556 573 Alexa Fluor 568 578 603 Alexa Fluor 594 590 617Alexa Fluor 633 621 639 Alexa Fluor 647 650 688 Alexa Fluor 660 663 690Alexa Fluor 680 679 702 Alexa Fluor 700 696 719 Alexa Fluor 750 752 779Cy2 489 506 Cy3 (512); 550 570; (615) Cy3,5 581 596; (640) Cy5 (625);650 670 Cy5,5 675 694 Cy7 743 767 Nucleic acid probes Hoeschst 33342 343483 DAP1 345 455 Hoechst 33258 345 478 SYTOX Blue 431 480 Chromomycin A3445 575 Mithramycin 445 575 YOYO-I 491 509 SYTOX Green 504 523 SYTOXOrange 547 570 Ethidium Bormide 493 620 7-AAD 546 647 Acridine Orange503 530/640 TOTO-1, TO-PRO-1 509 533 Thiazole Orange 510 530 PropidiumIodide (PI) 536 617 TOTO-3, TO-PRO-3 642 661 LDS 751 543; 590 712; 607Fluorescent Proteins Y66F 360 508 Y66H 360 442 EBFP 380 440 Wild-type396, 475 50, 503 GFPuv 385 508 ECFP 434 477 Y66W 436 485 S65A 471 504S65C 479 507 S65L 484 510 S65T 488 511 EGFP 489 508 EYFP 514 527 DsRed558 583 Other probes Monochlorobimane 380 461 Calcein 496 517 Ex: Peakexcitation wavelength (nm) Em: Peak emission wavelength (nm).

Any suitable method of analyzing fluorescence emission is encompassedherein. In this regard, techniques taught herein include but are notrestricted to 2-photon and 3-photon time resolved fluorescencespectroscopy as, for example, disclosed by Lakowicz et al. (1997)Biophys. J. 72:567, fluorescence lifetime imaging as, for example,disclosed by Eriksson et al. (1993) Biophys. J. 2:64 and fluorescenceresonance energy transfer as, for example, disclosed by Youvan et al.(1997) Biotechnology et elia 3: 1-18.

Luminescence and phosphorescence may result respectively from a suitableluminescent or phosphorescent label as is known in the art. Any opticalmeans of identifying such label may be used in this regard.

Infrared radiation may result from a suitable infrared dye. Exemplaryinfrared dyes that may be employed in the present disclosure include butare not limited to those disclosed in Lewis et al. (1999) Dyes Pigm.42(2): 197, Tawa et al. Mater. Res. Soc. Symp. Proc. 488 [Electrical,Optical and Magnetic Properties of Organic Solid-State Materials IV],885-890, Daneshvar et al. (1999) J. Immunol. Methods 226(1-2): 1 19-128,Rapaport et al. (1999) Appl. Phys. Lett. 74(3):329-33 \ and Durig et al.(1993) J Raman Spectrosc. 24(5):21-285. Any suitable infraredspectroscopic method may be employed to interrogate the infrared dye.For instance, fourier transform infrared spectroscopy as, for example,described by Rahman et al. (1998) J. Org. Chem. 63:6196 may be used inthis regard.

Suitably, electromagnetic scattering may result from diffraction,reflection, polarization or refraction of the incident electromagneticradiation including light and X-rays. Such scattering can be used toquantitate the level of mRNA or level of protein.

Flow cytometry is particularly useful in analyzing fluorophore emission.

As is known in the art, flow cytometry is a high throughput techniquewhich involves rapidly analyzing the physical and chemicalcharacteristics of particles (e.g. labeled mRNA, DNA or proteins) asthey pass through the path of one or more laser beams while suspended ina fluid stream. As each particle intercepts the laser beam, thescattered light and fluorescent light emitted by each cell or particleis detected and recorded using any suitable tracking algorithm as, forexample, described hereunder.

A modern flow cytometer is able to perform these tasks up to 100,000cells/particles s⁻¹. Through the use of an optical array of filters anddichroic mirrors, different wavelengths of fluorescent light can beseparated and simultaneously detected. In addition, a number of laserswith different excitation wavelengths may be used. Hence, a variety offluorophores can be used to target and examine, for example, differentimmune effectors within a sample or immune effectors from multiplesubjects.

Suitable flow cytometers which may be used in the methods of the presentdisclosure include those which measure five to nine optical parameters(see Table 2) using a single excitation laser, commonly an argon ionair-cooled laser operating at 15 mW on its 488 nm spectral line. Moreadvanced flow cytometers are capable of using multiple excitation laserssuch as a HeNe laser (633 nm) or a HeCd laser (325 nm) in addition tothe argon ion laser (488 or 514 nm).

TABLE 2 Exemplary optical parameters which may be measured by a flowcytometer. Detection angle form Wavelength Parameter Acronym incidentlaser beam (nm) Forward scattered light FS 2-5° 488

Side scattered light SS 90° 488

“Green” fluorescence FL1 90° 510-540^(†) “Yellow” fluorescence FL2 90°560-580^(†) “Red” fluorescence FL3 90° >650

  using a 488 nm excitation laser ^(†)width of bandpass filter ″longpassfilter

indicates data missing or illegible when filed

For example, Biggs et al. (1999) Cytometry 36:36-45 have constructed an1 1-parameter flow cytometer using three excitation lasers and havedemonstrated the use of nine distinguishable fluorophores in addition toforward and side scatter measurements for purposes of immunophenotyping(i.e. classifying) particles. Selection of parameters can be adequatelyused depends heavily on the extinction coefficients, quantum yields andamount of spectral overlap between all fluorophores (Malemed et al.(1990) “Flow cytometry and sorting”, 2^(nd) Ed., New York, Wiley-Liss).It will be understood that the present disclosure is not restricted toany particular flow cytometer or any particular set of parameters. Inthis regard, the disclosure also contemplates use in place of aconventional flow cytometer, a microfabricated flow cytometer as, forexample, disclosed by Fu et al. (1999) Nature Biotechnology 17: 1 109-11 1 1.

The assay enabled herein may be automated or semi-automated for highthroughput screening or for screening for a number of immune effectorsfrom the one subject. The automation is conveniently controlled bycomputer software.

The present disclosure further contemplates therefore web-based andnon-web-based systems where data on the cell-mediatedimmunoresponsiveness of a subject are provided by a client server orother architecture platform to a central processor which analyses andcompares to a control and optionally considers other information such aspatient age, sex, weight and other medical conditions and then providesa report, such as, for example, a risk factor for disease severity orprogression or status or an index of probability of disease development.A business method is therefore also provided whereby blood is collectedin transportable tubes which is then analyzed for cell-mediatedimmunoresponsiveness at a defined location and the results then sent inthe form of an electronic report via a client server or otherarchitecture platform to a clinical care provider.

Hence, knowledge-based computer software and hardware also form part ofthe present disclosure. This facilitates clinical care to ascertainwhether a disease condition including infection, cancer of inflammationor a medicament or toxicant is inducing or is associated withimmunosuppression.

The assays enabled by the instant disclosure may be used in existing ornewly developed, knowledge-based architecture or platforms associatedwith pathology services. For example, results from the assays aretransmitted via a communications network (e.g. the internet) ortelephone connection to a processing system in which an algorithm isstored and used to generate a predicted posterior probability valuewhich translates to the index of cell-mediated immunoresponsiveness orimmunosuppression which is then forwarded to an end user in the form ofa diagnostic or predictive report. This report may also form the basisof clinical care management and personalized medicine.

The assay may, therefore, be in the form of a kit or computer-basedsystem which comprises the reagents necessary to detect theconcentration of the immune effector molecule following exposure oflymphocytes to an inventive composition and the computer hardware and/orsoftware to facilitate determination and transmission of reports to aclinician.

For example, the present disclosure contemplates a method of allowing auser to determine the status of cell-mediated immunoresponsiveness of asubject, the method including:

(a) receiving data in the form of levels or concentrations of an immuneeffector molecule which, relative to a control, provide a correlation asthe state of cell-mediated immunoresponsiveness in a subject, via acommunications network, the immune effector molecule measured afterexposure of lymphocytes to an inventive composition;

(b) processing the subject data via univariate or multivariate analysisto provide an immunoresponsiveness value;

(c) determining the status of the subject in accordance with the resultsof the immunoresponsiveness value in comparison with predeterminedvalues; and

(d) transferring an indication of the status of the subject to the uservia the communications network.

Reference to the “univariate” or “multivariate” analysis includes analgorithm which performs the univariate or multivariate analysisfunction.

Conveniently, the method generally further includes:

(a) having the user determine the data using a remote end station; and

(b) transferring the data from the end station to the base station viathe communications network.

The base station can include first and second processing systems, inwhich case the method can include:

(a) transferring the data to the first processing system;

(b) transferring the data to the second processing system; and

(c) causing the first processing system to perform the univariate ormultivariate analysis function to generate the cell-mediatedimmunoresponsiveness value.

The method may also include:

(a) transferring the results of the univariate or multivariate analysisfunction to the first processing system; and

(b) causing the first processing system to determine the status of thesubject.

In this case, the method also includes at least one of:

(a) transferring the data between the communications network and thefirst processing system through a first firewall; and

(b) transferring the data between the first and the second processingsystems through a second firewall.

The second processing system may be coupled to a database adapted tostore predetermined data and/or the univariate or multivariate analysisfunction, the method including:

(a) querying the database to obtain at least selected predetermined dataor access to the univariate or multivariate analysis function from thedatabase; and

(b) comparing the selected predetermined data to the subject data orgenerating a predicted probability index of a level of cellularimmunoresponsiveness or immunosuppression.

The second processing system can be coupled to a database, the methodincluding storing the data in the database.

The method can also include causing the base station to:

(a) determine payment information, the payment information representingthe provision of payment by the user; and

(b) perform the comparison in response to the determination of thepayment information.

The present disclosure also provides a base station for determining thestatus of a subject with respect to cell-mediated immunoresponsivenessor immunosuppression, the base station including:

(a) a store method;

(b) a processing system, the processing system being adapted to:

(c) receive subject data from the user via a communications network, thedata including levels of immune effector molecule wherein the level ofthe effector molecule relative to a control provides a correlation tothe state of cell-mediated immunoresponsiveness wherein the immuneeffector molecule is determined after exposure of lymphocytes to aninventive composition;

(d) performing an algorithmic function including comparing the data topredetermined data;

(e) determining the status of the subject in accordance with the resultsof the algorithmic function including the comparison; and

(c) output an indication of the status of the subject to the user viathe communications network.

The processing system can be adapted to receive data from a remote endstation adapted to determine the data.

The processing system may include:

(a) a first processing system adapted to:

(i) receive the data; and

(ii) determine the status of the subject in accordance with the resultsof the univariate or multivariate analysis function including comparingthe data; and

(b) a second processing system adapted to:

(i) receive the data from the processing system;

(ii) perform the univariate or multivariate analysis function includingthe comparison; and

(iii) transfer the results to the first processing system.

The processing system can be coupled to a database, the processingsystem being adapted to store the data in the database.

In accordance with this embodiment, levels of the immune effectormolecule may be screened alone or in combination with other biomarkersor disease indicators. An “altered” level means an increase or elevationor a decrease or reduction in the concentrations of the immune effectormolecule.

The determination of the concentrations or levels of the immune effectormolecule enables establishment of a diagnostic rule based on theconcentrations relative to controls. Alternatively, the diagnostic ruleis based on the application of a statistical and machine learningalgorithm. Such an algorithm uses relationships between effectormolecule and disease status observed in training data (with knowndisease or cell-mediated immunoresponsiveness status) to inferrelationships which are then used to predict the status of subjects withunknown status. An algorithm can be employed which provides an index ofprobability that a subject has a certain level of cell-mediatedimmunoresponsiveness and/or a disease condition. The algorithm performsa univariate or multivariate analysis function.

Hence, the present disclosure provides a diagnostic rule based on theapplication of statistical and machine learning algorithms. Such analgorithm uses the relationships between immune effector molecule andlevel of cell-mediated immunoresponsiveness or immunosuppressionobserved in training data (with known immune status) to inferrelationships which are then used to predict the status of patients withunknown immune status. Practitioners skilled in the art of data analysisrecognize that many different forms of inferring relationships in thetraining data may be used without materially changing the presentdisclosure.

The present disclosure further contemplates the use of a knowledge baseof training data comprising levels of immune effector molecule from asubject with a known cell-mediated immunoresponsiveness level togenerate an algorithm which, upon input of a second knowledge base ofdata comprising levels of the same immune effector molecule from asubject with an unknown immunoresponsiveness level, provides an index ofprobability that predicts the nature of the cell-mediatedimmunoresponsiveness.

The term “training data” includes knowledge of levels of immune effectormolecule relative to a control wherein the immune effector molecule isdetermined after exposure of lymphocytes to an inventive composition. A“control” includes a comparison to levels of immune effector molecule ina subject with “normal” immurioresponsiveness or may be a statisticallydetermined level based on trials.

Hence, the term “training data” includes levels of an immune effectormolecule.

The levels or concentrations of the immune effector molecule provide theinput test data referred to herein as a “second knowledge base of data”.The second knowledge base of data either is considered relative to acontrol or is fed into an algorithm generated by a “first knowledge baseof data” which comprise information of the levels of an immune effectorin a subject with a known immunological status. The second knowledgebase of data is from a subject of unknown status with respect to cellmediated immunoresponsiveness. The output of the algorithm or thecomparison to a control is a probability or risk factor, referred toherein as “an index of probability”, of a subject having a certain levelof immunoresponsiveness or immunosuppressive.

Data generated from the levels of immune effector molecule are inputdata. The input of data comprising the immune effector levels iscompared with a control or is put into the algorithm which provides arisk value of the likelihood that the subject has, for example, animmunosuppressive condition. A treatment regime can also be monitored toascertain the presence of any immunosuppression. A level ofimmunosuppression may increase the risk of a subject getting a secondaryinfection or having a relapse (e.g. during cancer therapy or treatmentof a pathogenic infection).

As described above, methods for diagnosing an immunoresponsiveness orimmunosuppressive condition by determining the extent to which a subjectcan mount an innate and/or adaptive immune response via a level of animmune effector molecule provides a second knowledge base data in analgorithm generated with first knowledge base data or levels of the sameeffector molecule in subjects with a known immune status. Also providedare methods of detecting immunoresponsiveness comprising determining thepresence and/or velocity of an immune effector molecule followingstimulation of the innate and/or adaptive immune system in a subject'ssample. By “velocity” it is meant the change in the concentration of theeffector molecule in a subject's sample over time.

As indicated above, the term “sample” as used herein means any samplecontaining one or more lymphocytes including, but not limited to, wholeblood, a whole blood fraction, tissue extracts and freshly harvestedcells.

The method of the subject disclosure may be used in the diagnosis andstaging of a disease. The present disclosure may also be used to monitorthe progression of a condition and to monitor whether a particulartreatment is effective or not. In particular, the method can be used tomonitor immunosuppression following surgery, cancer therapy or other ormedication or exposure to toxicants.

In an embodiment, the subject disclosure contemplates a method formonitoring for immunosuppression in a subject, comprising:

(a) providing a sample from a subject;

(b) determining the level of an immune effector molecule followingstimulation by an inventive composition;

wherein the level of the immune effector relative to a control providesa correlation to the state of cell-mediated immunoresponsiveness andsubjecting the levels to an algorithm to provide an index of probabilityof the subject having a certain level of immunoresponsiveness; and

(c) repeating steps (a) and (b) at a later point in time and comparingthe result of step (b) with the result of step (c) wherein a differencein the index of probability is indicative of the progression of thecondition in the subject.

Reference to an “algorithm” or “algorithmic functions” as outlined aboveincludes the performance of a univariate or multivariate analysisfunction. A range of different architectures and platforms may beimplemented in addition to those described above. It will be appreciatedthat any form of architecture suitable for implementing the presentdisclosure may be used. However, one beneficial technique is the use ofdistributed architectures. In particular, a number of end stations maybe provided at respective geographical locations. This can increase theefficiency of the system by reducing data bandwidth costs andrequirements, as well as ensuring that if one base station becomescongested or a fault occurs, other end stations could take over. Thisalso allows load sharing or the like, to ensure access to the system isavailable at all times.

In this case, it would be necessary to ensure that the base stationcontains the same information and signature such that different endstations can be used.

It will also be appreciated that in one example, the end stations can behand-held devices, such as PDAs, mobile phones, or the like, which arecapable of transferring the subject data to the base station via acommunications network such as the Internet, arid receiving the reports.

In the above aspects, the term “data” means the levels or concentrationsof the immune effector following stimulation by a series of overlappingpeptides from about 7 to 14 amino acid residues in length whichencompass the entire length of a protein antigen. The “communicationsnetwork” includes the internet and mobile telephone network andtelephone land line. When a server is used, it is generally a clientserver or more particularly a simple object application protocol (SOAP).

One aspect of the present disclosure includes experiments thatdemonstrate the cell-mediated immune responsiveness of a subject bymeasuring responsiveness to an inventive composition. In an embodiment,one or more samples such as a sample of peripheral blood, of enrichedwhite

cell fraction of blood or bronchoalveolar lavage may be obtained from asubject having or suspected of development of a particular disease (e.g.autoimmune disease, infection to a pathogenic agent or exposure to aproteinaceous toxicant) and the immune responsiveness measured bydetermination of effector molecules from effector T-cells (e.g. CD4⁺T-cells and CD8⁺ T-cells).

The immunobinding methods include methods for detecting or quantifyingthe amount of a reactive component in a sample, which methods requirethe detection or quantitation of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containinga cytokine following stimulation of lymphocytes by an inventivecomposition and contacting the sample with an antibody and thendetecting or quantifying the amount of immune complexes formed under thespecific conditions.

Contacting the chosen biological sample with the antibody underconditions effective and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of adding the composition to the sample and incubating themixture for a period of time long enough for the antibodies to formimmune complexes with, i.e. to bind to, any effector molecules present.After this time, the sample-antibody composition, such as a tissuesection, ELISA plate, ELISpot, dot blot or Western blot, will generallybe washed to remove any non-specifically bound antibody species,allowing only those antibodies specifically bound within the primaryimmune complexes to be detected.

In a particular embodiment, the present disclosure teaches a method fordetecting the presence, absence, level or stage of a disease orcondition in a human subject, the method comprising contacting wholeblood, which comprises at least 10% of the total volume in a reactionmixture, with an inventive composition and measuring the presence orelevation in the level of an immune effector molecule from T-cellswherein the presence or level of the immune effector molecule isindicative of the disease or condition.

In a further embodiment, the present disclosure enables kits for usewith the methods described above. In one embodiment, an immunodetectionkit is contemplated. In another embodiment, a kit for analysis of asample from a subject having or suspected of developing a metal orchemically-induced disease is contemplated. In a more particularembodiment, a kit for analysis of a sample from a subject having orsuspected of developing a disease is contemplated. In an embodiment, akit is for assessing the cell-mediated immune responsiveness of asubject before or after a disease state has developed or before or aftera subject is given a medicament or is exposed to a toxicant orpollutant. If an antigen is also employed, the kit may also comprise aparticular antigen.

The immunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with orlinked to the given antibody or antigen, and detectable labels that areassociated with or attached to a secondary binding ligand. Exemplarysecondary ligands are those secondary antibodies that have bindingaffinity for the first antibody or antigen, and secondary antibodiesthat have binding affinity for a human antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody or antigen, along witha third antibody that has binding affinity for the second antibody, thethird antibody being linked to a detectable label.

The kits may further comprise a suitably aliquoted composition ofantigen or effector molecule, whether labeled or unlabeled, as may beused to prepare a standard curve for a detection assay.

The kits may contain antibody-label conjugates either in fullyconjugated form, in the form of intermediates, or as separate moietiesto be conjugated by the user of the kit. The components of the kits maybe packaged either in aqueous media or in lyophilized form.

The container means of any of the kits generally includes at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the testing agent, the antibody or antigen may be placed, andgenerally, suitably aliquoted. Where a second or third binding ligand oradditional component is provided, the kit will also generally contain asecond, third or other additional container into which this ligand orcomponent may be placed. The kits taught by the present disclosure alsotypically include a means for containing the antibody, peptides derivedfrom an antigen and any other reagent containers in close confinementfor commercial sale. Such containers may include injection orblow-molded plastic containers into which the desired vials areretained.

Also contemplated herein is an improved assay to detect a cell-mediatedimmune response or the level thereof in a subject, the assay comprisingincubating lymphocytes from the subject with an antigen and detectingfor the presence of or elevation in effector molecules, the improvementcomprising incubating the lymphocytes with an inventive composition.

The present disclosure further provides a method of treatment of asubject having a pathogenic infection, an autoimmune disorder or canceror a propensity for developing such a condition or disorder, the methodcomprising contacting a source of lymphocytes from the subject with aninventive composition and measuring the presence or elevation in thelevel of an immune effector molecule from T-cells wherein the presenceor level of the immune

effector molecule is indicative of the level of cell-mediatedresponsiveness of the subject which is indicative of the presence,absence, level or state of the condition or disorder and then treatingthe condition or disorder.

BIBLIOGRAPHY

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1. A composition comprising at least one fragment of ESAT-6 and at leastone fragment of CFP-10.
 2. The composition of claim 1, wherein the atleast one fragments of ESAT-6 and the at least one fragment of CFP-10comprise at least two sets of peptides, a first set comprising at leastone peptide of from about 7 to 14 amino acid residues in length and asecond set comprising at least one peptide of from 16 amino acidresidues or greater.
 3. The composition of claim 2, wherein the at leastone peptide of from about 7 to 14 amino acid residues is recognized byCD8+ lymphocytes and the at least one peptide of from 16 amino acid orgreater peptides is recognized by CD4+ lymphocytes.
 4. The compositionof claim 1, further comprising at least one sugar.
 5. The compositionaccording to claim 4, wherein the at least one sugar is a non-reducingsugar, or wherein the at least one sugar is trehalose.
 6. A process formeasuring cell-mediated immune response activity in a subject, saidmethod comprising: contacting immune cells comprising lymphocytesobtained from the subject with the composition of claim 1; and measuringpresence or elevation in a level of an immune effector molecule producedby said immune cells, wherein the presence or level of the immuneeffector molecule is indicative of a level of cell-mediatedresponsiveness of the subject.
 7. The process of claim 6, wherein eitheror both of (i) the immune cells are present in a sample of undilutedwhole blood obtained from the subject, and (ii) the whole blood iscollected in a tube comprising heparin.
 8. The process claim 6, whereinthe immune effector molecule is a cytokine.
 9. The process of claim 6,wherein measuring comprises detecting the immune effectors molecule withantibodies specific for the immune effector molecule.
 10. The process ofclaim 6, wherein the subject has an infection by a pathogenic agentselected from Mycobacterium species, Staphylococcus species,Streptococcus species, Borrelia species, Escherichia coli, Salmonellaspecies, Clostridium species, Shigella species, Proteus species,Bacillus species, Herpes virus, Hepatitis B or C virus, and Human immunedeficiency virus (HIV), or a disease resulting therefrom.
 11. Theprocess of claim 6, wherein the subject has a disease condition which isan infection by Mycobacterium tuberculosis or tuberculosis (TB).
 12. Theprocess of claim 6, wherein magnitude of the level of the immuneeffector molecule that is produced by the immune cells correlates withone or more of a state, progression and severity of a disease condition.13. A method for a user to determine status of cell-mediatedimmunoresponsiveness of a subject, the method comprising: (a) receivingsubject data in the form of levels or concentrations of an immuneeffector molecule which, relative to a control level, provide acorrelation to a state of cell-mediated immunoresponsiveness in thesubject, from the user via a communications network, the immune effectormolecule level being measured after exposure of lymphocytes from thesubject to the composition of claim 1, (b) processing the subject datavia univariate or multivariate analysis to provide animmunoresponsiveness value; (c) determining cell-mediatedimmunoresponsiveness status of the subject in accordance with results ofthe immunoresponsiveness value in comparison with predetermined values;and (d) transferring an indication of the cell-mediatedimmunoresponsiveness status of the subject to the user via thecommunications network.
 14. The method of claim 6, wherein thecomposition is contacted with the immune cells comprising lymphocyteswith at least one sugar present, wherein the at least one sugar is anon-reducing sugar, or wherein the at least one sugar is trehalose. 15.(canceled)
 16. The process of claim wherein the subject is selected froma human, a non-human primate, a livestock animal, a laboratory testanimal, a companion animal, a camel, an avian species, a reptile, and anamphibian.
 17. The process of claim 8, wherein the cytokine is IFN-γ.18. The process of claim 9, wherein measuring comprises detecting theimmune effector molecule in an enzyme-linked immunosorbent assay(ELISA).